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Random mutagenesis and recombination of sam1 gene by integrating error-prone PCR with staggered extension process.
- Source :
-
Biotechnology letters [Biotechnol Lett] 2008 Jul; Vol. 30 (7), pp. 1227-32. Date of Electronic Publication: 2008 Mar 04. - Publication Year :
- 2008
-
Abstract
- An efficient method for creating a DNA library is presented in which gene mutagenesis and recombination can be introduced by integrating error-prone PCR with a staggered extension process in one test tube. In this process, less than 15 cycles of error-prone PCR are used to introduce random mutations. After precipitated and washed with ethanol solution, the error-prone PCR product is directly used both as template and primers in the following staggered extension process to introduce DNA recombination. The method was validated by using adenosyl-methionine (AdoMet) synthetase gene, sam1, as a model. The full-length target DNA fragment was available after a single round. After being selected with a competitive inhibitor, ethionine, a mutated gene was obtained that increased AdoMet accumulation in vivo by 56%.
- Subjects :
- Antimetabolites pharmacology
Ethionine pharmacology
Methionine Adenosyltransferase antagonists & inhibitors
Methionine Adenosyltransferase genetics
Mutation
Recombination, Genetic drug effects
S-Adenosylmethionine biosynthesis
S-Adenosylmethionine genetics
Saccharomyces cerevisiae genetics
Saccharomyces cerevisiae Proteins antagonists & inhibitors
Saccharomyces cerevisiae Proteins genetics
Directed Molecular Evolution methods
Methionine Adenosyltransferase biosynthesis
Mutagenesis
Polymerase Chain Reaction methods
Saccharomyces cerevisiae enzymology
Saccharomyces cerevisiae Proteins biosynthesis
Subjects
Details
- Language :
- English
- ISSN :
- 0141-5492
- Volume :
- 30
- Issue :
- 7
- Database :
- MEDLINE
- Journal :
- Biotechnology letters
- Publication Type :
- Academic Journal
- Accession number :
- 18317700
- Full Text :
- https://doi.org/10.1007/s10529-008-9674-9